Blue Lagoon Algae Improve Uneven Skin Pigmentation: Results from in vitro Studies and from a Monocentric, Randomized, Double-Blind, Vehicle-Controlled, Split-Face Study.

INTRODUCTION: Bathing in the Blue Lagoon (BL) in Iceland benefits patients with psoriasis. Accordingly, the BL water contains algae with biological activities that improve skin barrier function and affect T-cell responses relevant for psoriasis. Bathing in the BL is also becoming increasingly popular among healthy individuals and anecdotal evidence suggests positive effects on uneven skin pigmentation. OBJECTIVE: The aim of the study was to address the impact of BL algae on skin pigmentation. METHODS: In this work, in vitro gene expression studies in melanocytes and a noninvasive in vivo study were conducted. RESULTS: We here report that normal human epidermal melanocytes, which had been treated with nontoxic concentrations of BL algae, show a significantly reduced expression of α melanocyte-stimulating hormone-induced expression of genes important for melanin synthesis, such as tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, melan A protein, and pre-melanosome protein. This in vitro observation prompted us to conduct a randomized, double-blind, intra-individual, comparative split-face in vivo study, in which 60 volunteers with pre-existing facial pigment spots were treated twice daily with a BL algae containing serum or a vehicle control. We found that constitutive skin pigmentation as determined by colorimetry (individual typology angle and luminescence) did not differ significantly between vehicle- and serum-treated skin sites. In marked contrast, digital photography under cross-polarized lighting and RBX technology (VISIA CR) revealed that the number of pigment spots in the serum-treated face decreased significantly compared to the vehicle-treated side. CONCLUSION: Thus, BL algae can affect human melanocyte function in vitro and reduce uneven facial skin pigmentation in vivo.

Topical ceramides neither enhance UVB-induced apoptosis in normal human keratinocytes nor affect viability in UVB-irradiated reconstructed human epidermis.

Ceramides are the major lipid of lamellar sheets present in intercellular spaces of the stratum corneum contributing to epidermal barrier properties. Therefore, ceramides and their analogues have been studied for barrier enhancing and water-holding properties for decades. In vitro studies have indicated cytotoxic potential for cell-permeable ceramides thereby raising the question whether topical ceramide application might contribute to UVB-induced apoptosis. Phytosphingosine, N-hexanoyl-phytosphingosine and N-stearoylphytosphingosine (ceramide III) in concentrations ≤5 µm have been used for co-stimulation with low (160 J/m(2) ) or high (600 J/m(2) ) UVB doses in subconfluent basal and confluent differentiating keratinocytes. Significantly, increased caspase-3 activity was observed in basal keratinocytes irradiated with 600 J/m(2) UVB and in differentiating keratinocytes with both UVB doses. Co-stimulation with the named ceramides did not further increase (i) caspase-3 activity and (ii) nucleosomal fragmentation in differentiating keratinocytes. Moreover, co-stimulation with 1-mm ceramides did not further affect viability/lactate dehydrogenase release in UVB-irradiated reconstructed human epidermis corroborating the safety of these ceramides.

Modulation of skin pigmentation by the tetrapeptide PKEK: in vitro and in vivo evidence for skin whitening effects.

Uneven skin pigmentation is a significant cosmetic concern, and the identification of topically applicable molecules to address this issue is of general interest. We report that the tetrapeptide PKEK (Pro-Lys-Glu-Lys) can exert skin whitening effects based on one in vitro and four double-blinded vehicle-controlled in vivo studies. (i) Treatment of human keratinocytes with PKEK significantly reduced UVB-stimulated mRNA expression of interleukin (IL)-6, IL-8 and TNF-α and, most importantly, proopiomelanocorticotropin (POMC), i.e. a gene encoding the pigmentation-inducing soluble mediator α- (α-MSH). (ii) PKEK treatment significantly inhibited UVB-induced upregulation of genes encoding for IL-1α, IL-6, IL-8, TNF-α as well as POMC and tyrosinase in 10 healthy volunteers pretreated with PKEK for 4 weeks once daily. (iii) In a study enrolling 39 Caucasian women, facial pigment spots significantly faded after 6 weeks when PKEK was combined with the skin whitener sodium ascorbyl phosphate (SAP), whereas PKEK or SAP alone led to less pronounced fading of the pigment spots. (iv) Addition of PKEK enhanced the skin whitening potency of a SAP-containing preparation if applied for 8 weeks to the back of hands of 19 Caucasians. (v) 27 Japanese women were treated on their faces twice daily with an SAP only or a PKEK+SAP-containing formulation for 8 weeks. Application of PKEK+SAP significantly reduced skin pigmentation by 26% and by 18% according to SCINEXA score. We demonstrate that PKEK has the capacity to reduce UVB-induced skin pigmentation and may be suited to serve as a skin tone-modulating agent in cosmetic products.

Carbon nanoparticles induce ceramide- and lipid raft-dependent signalling in lung epithelial cells: a target for a preventive strategy against environmentally-induced lung inflammation.

BACKGROUND: Particulate air pollution in lung epithelial cells induces pathogenic endpoints like proliferation, apoptosis, and pro-inflammatory reactions. The activation of the epidermal growth factor receptor (EGFR) is a key event responsible for signalling events involving mitogen activated protein kinases specific for these endpoints. The molecular events leading to receptor activation however are not well understood. These events are relevant for the toxicological evaluation of inhalable particles as well as for potential preventive strategies in situations when particulate air pollution cannot be avoided. The current study therefore had the objective to elucidate membrane-coupled events leading to EGFR activation and the subsequent signalling cascade in lung epithelial cells. Furthermore, we aimed to identify the molecular target of ectoine, a biophysical active substance which we described to prevent carbon nanoparticle-induced lung inflammation. METHODS: Membrane signalling events were investigated in isolated lipid rafts from lung epithelial cells with regard to lipid and protein content of the signalling platforms. Using positive and negative intervention approaches, lipid raft changes, subsequent signalling events, and lung inflammation were investigated in vitro in lung epithelial cells (RLE-6TN) and in vivo in exposed animals. RESULTS: Carbon nanoparticle treatment specifically led to an accumulation of ceramides in lipid rafts. Detailed analyses demonstrated a causal link of ceramides and subsequent EGFR activation coupled with a loss of the receptor in the lipid raft fractions. In vitro and in vivo investigations demonstrate the relevance of these events for carbon nanoparticle-induced lung inflammation. Moreover, the compatible solute ectoine was able to prevent ceramide-mediated EGFR phosphorylation and subsequent signalling as well as lung inflammation in vivo. CONCLUSION: The data identify a so far unknown event in pro-inflammatory signalling and contribute to the understanding of particle cell interaction and therefore to risk identification and risk assessment of inhalable xenobiotics. Moreover, as this cellular reaction can be prevented by the well tolerated substance ectoine, a molecular preventive strategy for susceptible persons against airway inflammation is proposed.

Photoprotection against UVAR: effective triterpenoids require a lipid raft stabilizing chemical structure.

UVA(Ultraviolet A)-induced gene expression is supposed to be a hallmark for inflammation, for immunosuppression and in long-term cancer formation. In previous studies, we have shown for keratinocytes that physiological doses of UVA radiation result in the upregulation of gene expression mediated by ceramide formation from sphingolipids/cholesterol-rich microdomains (rafts), which can be blocked by preloading keratinocytes with cholesterol or plant sterols. Here, we show that besides stigmasterol and ß-sitosterol, also sterols like 14-dehydroergosterol, ergosterol-peroxide and 29-norcycloartenol inhibit the UVA response. Moreover, we present evidence that natural material-derived triterpenoids such as oleanolic acid can abrogate UVA-induced gene expression by raft stabilization. This effect depends on the structure of the molecule, because its isomer ursolic acid also integrates within the rafts without inhibiting ceramide formation and upregulation of gene expression.

Bioactive tetrapeptide GEKG boosts extracellular matrix formation: in vitro and in vivo molecular and clinical proof.

The 'matrikine' concept claims that processing of the precursors for collagen results in the formation of peptides such as KTTKS which in turn augments extracellular matrix (ECM) production. In the present study, we show the development of an anti-ageing active from an in silico approach by molecular design resulting in the tetrapeptide GEKG derived from ECM proteins. The efficacy of the peptide to significantly induce collagen production of the protein level and mRNA level has been demonstrated in vitro in human dermal fibroblasts and in vivo in a double-blind, randomized, placebo-controlled study enroling 10 volunteers with an average age of 48.2 years. The effect of GEKG on facial wrinkles was studied in 30 volunteers using state of the art fringe projection, which allows determination of surface roughness in three-dimensions. Here, only GEKG but not the placebo was able to significantly decrease skin roughness as a measure for wrinkles.

Ultraviolet A induces transport of compatible organic osmolytes in human dermal fibroblasts.

Compatible organic osmolytes, such as betaine, myo-inositol and taurine, are involved in cell protection. Human dermal fibroblasts accumulate these osmolytes and express mRNA specific for their transporting systems betaine-/gamma-amino-n-butyric acid (GABA) transporter (BGT-1), sodium-dependent myo-inositol transporter (SMIT) and taurine transporter (TAUT). Taurine uptake was about sixfold higher than that of betaine and myo-inositol. Compared with normoosmotic (305 mOsm/l) control, hyperosmotic exposure (405 mOsm/l) led to a twofold induction of osmolyte uptake. Ultraviolet A (UVA) upregulated osmolyte transporter mRNA levels and increased osmolyte uptake. Taurine inhibited UVA-induced interleukin-6 (Il-6) mRNA expression by 40%. Furthermore, Il-6 accumulation in the supernatants of UVA-irradiated dermal fibroblasts was much slower when cells were preincubated with taurine. These data indicate that taurine accumulation seems to be part of the fibroblast response to UVA radiation and may protect against UVA-induced Il-6 overexpression.

Bioactive molecules from the Blue Lagoon: in vitro and in vivo assessment of silica mud and microalgae extracts for their effects on skin barrier function and prevention of skin ageing.

Bathing in the Blue Lagoon, a specific geothermal biotope in Iceland has been known for many years to be beneficial for human skin in general and for patients with psoriasis and atopic dermatitis in particular. The scientific rationale for this empirical observation, however has remained elusive. We now report that extracts prepared from silica mud and two different microalgae species derived from the Blue Lagoon are capable of inducing involucrin, loricrin, transglutaminase-1 and filaggrin gene expression in primary human epidermal keratinocytes. The same extracts also affects primary human dermal fibroblasts, because extracts from silica mud and one type of algae inhibited UVA radiation-induced upregulation of matrix metalloproteinase-1 expression and both algae, as well as silica mud extracts induced collagen 1A1 and 1A2 gene expression in this cell type. These effects were not restricted to the in vitro situation because topical treatment of healthy human skin (n = 20) with a galenic formulation containing all three extracts induced identical gene regulatory effects in vivo, which were associated with a significant reduction of transepidermal water loss. In aggregate, these results suggest that the bioactives in Blue Lagoon have the capacity to improve skin barrier function and to prevent premature skin ageing. These observations explain at least some of the beneficial effects of bathing in the Blue Lagoon and provide a scientific basis for the use of Blue Lagoon extracts in cosmetic and/or medical products.

MicroRNA-15b regulates mitochondrial ROS production and the senescence-associated secretory phenotype through sirtuin 4/SIRT4.

Mammalian sirtuins are involved in the control of metabolism and life-span regulation. Here, we link the mitochondrial sirtuin SIRT4 with cellular senescence, skin aging, and mitochondrial dysfunction. SIRT4 expression significantly increased in human dermal fibroblasts undergoing replicative or stress-induced senescence triggered by UVB or gamma-irradiation. In-vivo, SIRT4 mRNA levels were upregulated in photoaged vs. non-photoaged human skin. Interestingly, in all models of cellular senescence and in photoaged skin, upregulation of SIRT4 expression was associated with decreased levels of miR-15b. The latter was causally linked to increased SIRT4 expression because miR-15b targets a functional binding site in the SIRT4 gene and transfection of oligonucleotides mimicking miR-15b function prevented SIRT4 upregulation in senescent cells. Importantly, increased SIRT4 negatively impacted on mitochondrial functions and contributed to the development of a senescent phenotype. Accordingly, we observed that inhibition of miR-15b, in a SIRT4-dependent manner, increased generation of mitochondrial reactive oxygen species, decreased mitochondrial membrane potential, and modulated mRNA levels of nuclear encoded mitochondrial genes and components of the senescence-associated secretory phenotype (SASP). Thus, miR-15b is a negative regulator of stress-induced SIRT4 expression thereby counteracting senescence associated mitochondrial dysfunction and regulating the SASP and possibly organ aging, such as photoaging of human skin.

Ultraviolet A-induced signaling involves a ceramide-mediated autocrine loop leading to ceramide de novo synthesis.

Exposure of human keratinocytes to ultraviolet A (UVA) radiation at physiological doses leads to a biphasic activation of transcription factor activator protein-2 (AP-2) and subsequently to a biphasic increase in gene expression of, e.g. intercellular adhesion molecule-1 (ICAM-1). Both kinetics follow a pattern with a first peak between 0.5 and 2 h and a second, more sustained activation between 16 and 48 h. We have previously reported on a non-enzymatic triggering of the ceramide signaling cascade as the initiating step in UVA radiation-induced signaling. In this study, we report that this early (0.5-1 h) peak in ceramide content is followed by a second peak that (i) was associated with an increased expression and activity of serine palmitoyltransferase, the key enzyme of ceramide synthesis, (ii) could be prevented by inhibitors of this enzyme, and (iii) was of functional relevance because its inhibition abrogated the second, but not the first peak in UVA radiation-induced ICAM-1 gene expression. We hypothesize that this second peak most likely resulted from a ceramide-mediated autocrine loop, for (i) inhibition of the first ceramide peak resulted in inhibition of the second peak and (ii) cell-permeable ceramides-induced serine palmitoyltransferase expression, activity, and subsequently ceramide content.

Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression.

Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (AMP; LL-37 and ß-defensin-2) expression. Urea stimulates the expression of, and is transported into, keratinocytes by two urea transporters (UTs), UT-A1 and UT-A2, and by aquaporins 3, 7, and 9. Inhibitors of these UTs block the downstream biological effects of urea, which include increased mRNA and protein levels of (i) transglutaminase-1, involucrin, loricrin, and filaggrin, (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and ß-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and AMP expression in a murine model of atopic dermatitis. Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and AMP expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin.

Mitochondrial cytochrome c release mediates ceramide-induced activator protein 2 activation and gene expression in keratinocytes.

The intracellular signaling pathway(s) through which second messenger ceramides induce gene expression in human cells has not yet been characterized. In the present study, ceramide-induced expression of intercellular adhesion molecule-1 (ICAM-1), which requires activation of transcription factor activator protein 2 (AP-2), was found to be mediated through a mitochondrial pathway. Inhibitors of mitochondrial electron transport chain (e.g. rotenone, thenoyltrifluoroacetone, and antimycin A) reduced ceramide-induced ICAM-1 expression. Stimulation of human keratinocytes with cell-permeant ceramides at concentrations that did not induce apoptosis (no activation of caspases 3, 8, and 9 and no nucleosomal fragmentation) but caused AP-2 activation and ICAM-1 induction released cytochrome c (cyt c) from mitochondria into the cytoplasm of cells. This cyt c release was an indispensable prerequisite for effective ceramide signaling, because its inhibition by modulating the mitochondrial megachannel with bonkrekic acid or carboxyatractyloside prevented ceramide-induced AP-2 activation and ICAM-1 expression. Analysis of the interaction between cyt c and AP-2 revealed that cyt c oxidized AP-2 and that this redox regulation greatly enhanced the DNA binding capacity of AP-2. Mitochondria thus have a previously unrecognized function in signaling ceramide-induced transcription factor activation and gene regulation.